Yeast Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes

Glutathione (GSH), a tripeptide composed of glycine, cysteine, and glutamic acid, is an abundant thiol found in a wide variety of cells, ranging from bacterial to mammalian cells. Adequate levels of GSH are essential for maintaining iron homeostasis. The ratio of oxidized/reduced GSH is strictly regulated in each organelle to maintain the cellular redox potential. Cellular redox imbalances cause defects in physiological activities, which can lead to various diseases. Although there are many reports regarding the cellular response to GSH depletion, studies on stress response to high levels of GSH are limited. Here, we performed genome-scale screening in the yeast Saccharomyces cerevisiae and identified RIM11, BMH1, and WHI2 as multicopy suppressors of the growth defect caused by GSH stress. The deletion strains of each gene were sensitive to GSH. We found that Rim11, a kinase important in the regulation of meiosis, was activated via autophosphorylation upon GSH stress in a glucose-rich medium. Furthermore, RNA-seq revealed that transcription of phospholipid biosynthetic genes was downregulated under GSH stress, and introduction of multiple copies of RIM11 counteracted this effect. These results demonstrate that S. cerevisiae copes with GSH stress via multiple stress-responsive pathways, including a part of the adaptive pathway to glucose limitation.

Reviewer #2 (Remarks to the Author): Yasukawa et al performed a screen designed to isolate genes that modulate the growth defect in S. cerevisiae associated with the uptake of high level of glutathione from the medium (facilitated by high-level expression of the plasma membrane glutathione transporter).They describe the isolation of three multicopy suppressors (RIM11, BHM1, and WHI2).Initially the authors focus on how loss or overexpression of these three genes impacts the ability of cells to survive under conditions of increased glutathione uptake.Later, the authors focus on Rim11 and explore how Rim11 kinase activity alters transcriptional profiling connected with the exposure to GSH.The authors propose a model for the upregulation of a subset of genes involved in lipid biogenesis that is dependent on Rim11 kinase activity and occurs in response to the cellular uptake of exogenous glutathione.The authors go on to make a series of observations about various players connected to Rim11 signaling and/or the transcription of lipid biogenesis genes, including OPI1, UME6, IME1, and INO1.They report transcriptional changes observed in the presence of GSH, as well as growth differences in the presence, absence, or overexpression of these genes.
Overall, the manuscript contains several interesting observations that suggest unexpected signaling activated in the presence of exogenous GSH, a less explored "stressor".Yet, in its current form, the manuscript feels incomplete in its goal to outline a clear Rim11-dependent signaling output that is associated with GSH uptake.While the authors outline a model in Figure 7, this model relies heavily on relationships anticipated based on prior studies focused on the signaling outlined in a variety of conditions, not necessarily connections established by the authors to occur as part of GSH-induced signaling.In broad terms, it feels like a significant jump from the reported data to the model of Figure 7, including proteins in the model that are not discussed or explored in the manuscript.The extent of the experiments do not seem to match with the overall conclusions and model.Some suggestions as to further studies that may help to focus or complete the study: 1. Arguably, the manuscript could benefit from some reorganization, with a clear focus from the initial results on Rim11.For example, reporting the initial isolation of RIM11 as a high-copy suppressor, and a straightforward analysis of whether overexpression of RIM11 requires Ire1 to confer resistance to GSH in the HGT1 strain, would streamline the results.These straightforward experiments would establish a role for Rim11, and a role that is outside the prior focus on the UPR that the Toledano work reported in Kumar et al 2011.
2. As the manuscript is currently setup, the authors make epistasis arguments about the three initially isolated suppressors from the spotting assay in Figure 1E.These conclusions appear to be a potential overinterpretation of the data.It is also unclear what the pathway organization (if true) means in the context of the later analysis of, and focus on, Rim11.
The growth defects observed between the three individual mutants on plates are modest and seem variable (Fig 1D the bhm1∆ and whi2∆ growth are called similar, versus in Fig 1E the bmh1∆ shows better growth than the whi2∆).This growth variability makes the argument as to whether there is a true additive impact, or if the difference in growth is just assay-to-assay variation, difficult to discern.Similarly, the growth suppression of the null strains with the various 2µ plasmids is suggestive, but not particularly robust or fully consistent with the outlined model.It is unclear in the outlined epistasis diagram why a bhm1∆ would not be bypassed by additional RIM11 (?) If the authors do want to make arguments about epistasis, some evidence of reproducibility is also warranted, including how many replicates were performed and/or showing replicates of the strains.
3. The authors do not validate the transcriptional upregulation they outline for their RNA-seq dataset.Some validation by RT-PCR would strengthen the conclusions.It would also allow one to establish if Rim11 is required for the upregulation (e.g., analysis in a rim11∆).Such data would complement the focus on growth as the major determinant for a signaling role.
4. The experiments of Figure 6 exploring Opi1, Ime1, Ume6 and Ino1 expression or localization during conditions of increased GSH are suggestive of unique roles.However, these data could be more robust.The variable expression of Opi1 in the "GSH stress" cells makes the co-localization data difficult to parse.It is not clear why a more intense western band for Ume6-FLAG implies an increased in "Ume6 phosphorylation".
5. The authors observe and say that the degree and/or location of phosphorylation on Rim11 is likely distinct for GSH and glucose starvation, but yet then the authors argue that GSH induces a glucose starvation response (in the discussion).If the authors wish to imply that glucose transporters, etc. are important players in the response to GSH (as discussed), then some experiments to this point should be performed.Or at a minimum, the transcriptional signature that the authors use in the discussion to support a glucose starvation response should be more clearly outlined in the results.results reported previously" of Kumar et al 2011.It seems a missed opportunity for a more descriptive comparison.Given the prior work followed the transcriptional response at an earlier and lower dose (50 µM GSH for 30 min), one would expect some differences.
7. Minor point.The authors convincingly establish that HGT1 overexpression confers sensitivity to exogenous GSH as reported previously.Yet, it is unclear what the % quantification of GSH (8-9% intracellular GSH concentration) refers to in Figure 1B.One presumes this is 8-9% of the total GSH added to the culture?
In this work, the authors examined the effects of high levels of glutathione (GSH) on the stress response in yeast.Through a genomic screen, the RIM11, BMH1, and WHI2 genes were identified as high copy number suppressors of the growth inhibition caused by GSH stress.Strains carrying mutations in each of these genes were sensitive to GSH.The study then focused on Rim11, the yeast homolog of glycogen synthase kinase 3beta.This protein kinase, which is subject to autophosphorylation, has been associated with the phosphorylation of transcription factors involved with meiosis, and a recent paper revealed that Rim11 regulates the partitioning of the lipid intermediate phosphatidate (PA) between the synthesis of membrane phospholipids and the neutral lipid triacylglycerol via the phosphorylation and inhibition of the PA phosphatase activity encoded by PAH1 (Khondker et al. (2022).Here, the authors showed through an RNA-seq analysis that RNA levels of UAS INO1,CHO1,CHO2,OPI3,etc.) were repressed under GSH stress, whereas multiple copies of RIM11 caused the derepression of these genes.The Rim11-mediated effects of the gene expression were dependent on its protein kinase activity.The work also demonstrated that GSH invokes multiple stress-responsive pathways, including a part of the adaptive pathway to glucose limitation.
Overall, major conclusions are supported by a sound experimental approach.The data indicate that Rim11 protein kinase activity regulates transcription of UAS INO -containing phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit in response to GSH stress.Major shortcomings of the work, however, are that (1) expression of the encoded protein/phospholipid synthesizing activity of at least one gene identified by the RNA-seq analysis be confirmed and that (2) the cellular phospholipid content be correlated with the Rim11-mediated expression of the UAS INO -containing genes.
Response: Thank you for these helpful suggestions.We have included several new experiments to address the points raised, including points (1) and (2) that are mentioned above.We have addressed each of the specific comments below.
Although the title "Yeast Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes," the Introduction and Discussion pay little attention to the wealth of information about lipid synthesis, especially with regard to how the findings of this work relates to what is known about the Rim11-mediated regulation of lipid synthesis via the phosphorylation of Pah1.Pah1 is the PA phosphatase enzyme that regulates the levels of its substrate PA, which in turn governs the location and repressor function of Opi1.The authors show that Opi1 location is regulated by GSH stress and Rim11 protein kinase activity, but don't draw the connection between the Rim11-mediated phosphorylation of Pah1, the inhibition of its PA phosphatase activity, and regulation of lipid synthesis.Instead they focus on hypothetical scenarios for which clear cut evidence is missing (see specific comments below).Khondker et al. (2022) (briefly mentioned in passing at the end of the Discussion) showed that Rim11, expressed and purified from yeast where it is subject to its autophosphorylation, phosphorylates Pah1 on multiple residues .Pah1 is the PA phosphatase that dephosphorylates PA to produce diacylglycerol, which is then acylated to form triacylglycerol.Mutants lacking PA phosphatase activity accumulate PA, which cause the retention of Opi1 outside the nucleus resulting in the derepression of the UAS INO -containing genes (especially CHO1 (ref 15 in Khondker et al 2022) and an increase in the synthesis of phospholipids (Also see ref.Overall, the impact of this work with respect to lipid synthesis is high, but the authors are not seeing it because they are generally unaware of the crucial role Pah1 PA phosphatase plays in the regulation of PA levels and lipid synthesis, and that PA phosphatase is regulated by phosphorylation via Rim11.
Response: Regarding the relationship between the GSH stress response and phospholipid biosynthesis, we have added the following sentences in the Discussion.We have comprehensively discussed the relationships between GSH treatment, Rim11, and Pah1.
(Page 14; line 20-27, in the revised manuscript) "Figure 8A shows the function of Rim11 in the GSH stress response.Khondker et al. (2022) reported that Rim11 phosphorylates phosphatidic acid phosphatase (Pah1), and thereby inhibits its phosphatase activity.Pah1 is dephosphorylated by the Nem1-Spo7 complex and is subsequently recruited to the nuclear/ER membranes where it performs its functions (Karanasios et al., 2010;Choi, et al., 2011).At the membrane, Pah1 dephosphorylates phosphatidic acid and converts it into DAG.This regulates the localization and function of Opi1, which is tethered to the ER membrane via interaction with PA and the ER membrane protein, Scs2 (Loewen et al., 2004).Therefore, it is likely that the kinase activity of Rim11 and the function of Pah1 play an important role in the recovery of phospholipid biosynthetic gene expression levels in the GSH stress response."(Page 14 line 34-39, in the revised manuscript) "At the protein level, Ino1-GFP was significantly decreased upon GSH treatment (Figure 7A).Moreover, PC, PE, and PS contents were decreased upon GSH treatment (Figure 7D).We hypothesize that Rim11 translocates into the nucleus following GSH treatment (Figure 4C, Supplemental Fig. 4C), and phosphorylation of Pah1 by Rim11 is decreased.We predict that reactivated Pah1 converts PA to DAG, which leads to the reduction of PA levels and translocation of Opi1 into the nucleus, ultimately leading to repression of the transcription of the phospholipid biosynthetic gene (Figure 8B)." (Page 15 line 5-11, in the revised manuscript) "The amount of Ino1-GFP was consistent with the changes in the INO1 mRNA level (Figure 7A).Moreover, lipidomic analyses revealed that overexpression of RIM11 increased the overall lipid contents in the cell (Figure 7D).Therefore, we hypothesized that de-repression of the decreased biosynthetic activity of phospholipids by Rim11, and the increased expression of the INO1 gene, may contribute to enhanced tolerance of the cells to GSH.Specifically, overexpressed Rim11 phosphorylates Pah1, thereby inhibiting the dephosphorylating activity of Pah1.We hypothesize that the proportion of Opi1, which is anchored on the ER membrane, then increases, causing transcriptional de-repression of the phospholipid biosynthetic genes."

Specific comments:
Page 5 Line 30-the authors do not propose an explanation for why RIM11 under its native promoter did not rescue the growth phenotype.

Response:
We deduced that one of the possibilities was that the original promoter region was not sufficiently long.Therefore, a longer 5'-region was subcloned as a promoter of RIM11, and recovery of the GSH sensitivity was assessed.As shown in Figure for the reviewers A, phenotypic recovery was partial even with the longer 5'-region, suggesting that some other elements on the original plasmid obtained by the high-copy suppressor screening may be needed for sufficient expression of the RIM11 gene.Alternatively, as RIM11 expression increases in the stationary phase (Gasch et al., 2000), we speculate that it was only insufficiently expressed under the experimental conditions shown in Figure 1D.However, as this result does not seem to affect the main findings described in this manuscript, we only added a brief explanation to the main text.
We have added the following sentences in the main text: (Page 5; line 34-38, in the revised manuscript) "Although we also tested the long RIM11 5'-region as a promoter, no increase in growth phenotype rescue was observed (data not shown).It is possible that other elements present on the original plasmid obtained by the high-copy suppressor screening were needed for sufficient expression of the RIM11 gene.Alternatively, as expression of RIM11 increases in the stationary phase (Gasch et al., 2000), RIM11 may be insufficiently expressed by its own promoter under the conditions shown in Figure 1D."Response: We agree that there are some inconsistencies between the results and the model.We carefully reconsidered the results of the spot assays and concluded that it was difficult to create a diagram that reflected all the data correctly.Therefore, we have removed the diagram from the revised manuscript.Please also see our response to the next point.

Figure 1F
-There is no clear explanation in the text as to why there is no genetic interaction between Rim11 and Whi2.The proposed model also doesn't explain why overexpression of Rim11 rescues the growth defect in whi2Δ but not bmh1Δ, nor why Whi2 overexpression rescues rim11Δ.

Response:
We believe that genetic interaction occurred between rim11 and whi2.As shown in Figure 1E, the Δrim11 Δwhi2 strain showed higher GSH sensitivity than did the Δwhi2 strain.
Moreover, as shown in Figure 1F, overexpression of RIM11 partially rescued GSH sensitivity of the Δwhi2 strain, and vice versa.Overexpression of WHI2 partially rescued GSH sensitivity of the Δrim11 strain.As we mentioned above and as suggested by you, we concluded that it was difficult to create a diagram that reflected all the data correctly.Therefore, we have removed the diagram from the revised manuscript.To explain the results more clearly and accurately than in the original manuscript, we have revised the main text as follows: (Page 5-6, line 33-1, in the original manuscript) "We further examined the GSH stress sensitivity of double or triple mutants of these three genes (Figure 1E).The Δbmh1Δwhi2 and Δrim11Δbmh1 HGT1 strains showed augmented GSH stress sensitivity compared with their single-deletion mutants, and the latter strain showed less severe additive effects, indicating the existence of genetic interactions between BMH1 and WHI2, or BMH1 and RIM11.To investigate whether functional interactions exist among these three genes in the GSH stress response pathway, each single-deletion mutant was transformed with other suppressors (Figure 1F).The results suggested that BMH1 is located upstream of WHI2 and showed that loss of one of the two genes markedly decreased GSH stress tolerance.In the schematic diagram in Figure 1F, this relationship is depicted as "/" and the degree of importance of each signal is indicated with the thickness of the arrows."(Page 5-6, line 39-8, in the revised manuscript) "We further examined the GSH stress sensitivity of double or triple mutants of these three genes (Figure 1E).The Δbmh1Δwhi2 and Δrim11Δbmh1 HGT1 strains showed augmented GSH stress sensitivity compared with their single-deletion mutants, as shown in Fig. 1E.Furthermore, RIM11 overexpression did not rescue the GSH sensitivity of the Δbmh1 HGT1 strain, and BMH1 overexpression did not rescue the GSH sensitivity of Δrim11 HGT1 (Figure 1F).These results suggest that Bmh1 may function in a different pathway than Rim11.Overexpression of RIM11 partially rescued the GSH sensitivity of the Δwhi2 HGT1 strain.Also, overexpression of WHI2 rescued the GSH sensitivity of the Δrim11 HGT1 strain (Fig. 1F).These results suggest that Rim11 and Whi2 may function in the similar pathways.The Δwhi2Δrim11 HGT1 strain showed similar levels of sensitivity to Δrim11 HGT1 strain.In contrast, the Δwhi2Δrim11 HGT1 strain was more sensitive to GSH than the Δwhi2 HGT1 strain (Fig. 1E).These results may suggest Rim11 has more important role than Whi2 in response to GSH stress." Page 7 Line 20was there a specific reason the TDH3 promoter was used?
Response: Our intention with the use of the TDH3 promoter was to express the Rim11 protein at a high level.We integrated the cassette containing the ORF under the control of the TDH3 promoter and aimed to express it uniformly in all cells.Furthermore, the TDH3 promoter maintains relatively high activity even after glucose depletion (Partow et al., 2010, Yeast, 27: 955 -964).

Page 7 Line 30-32 -missing citation
Response: We have added the following reference in the main text.
(Page 8, line 1, in the revised manuscript) Response: We revised the sentence in the main text as follows: (Page 9, line 32-34, in the revised manuscript) "In contrast, we performed GO-based analysis for 41 commonly downregulated DEGs in kinase-dead datasets (i), (ii), and (iii) (Figure 5C) under the same conditions mentioned above.Among these, the 14 downregulated genes were categorized as being involved in the lipid biosynthetic process (GO:0008610) (Figure 5D)." The RNA-seq data would benefit from an examination of protein levels and lipid analysis to confirm the gene expression-enzyme product relationship.
Response: We selected INO1 among the genes that exhibited distinctive changes in expression between conditions and analyzed its protein levels.The Ino1-GFP expression construct was integrated into the chromosome of the HGT1 strain and Ino1-GFP was detected using Western blotting with an anti-GFP antibody.GSH treatment of the HGT1 Rim11 strain significantly decreased the Ino1-GFP protein levels, which was consistent with the change in INO1 transcription levels as revealed by RNA-seq analysis.This result is presented in Supplemental Figure 5B in the revised manuscript.Furthermore, in the presence of GSH, overexpression of RIM11 increased the Ino1-GFP protein levels by 2.5-fold, and overexpression of RIM1-K68A decreased it by 11-fold.This result is consistent with the RNA-seq data of INO1 in sample set (iv) and (iii), in which log 2 fc of HGT1 RIM11 OE / HGT1 RIM11 and of HGT1 RIM11-K68A OE / HGT1 RIM11 OE were 1.84 and -4.37, respectively (Figure 5D, Table 2).We included this data as Figure 7A in the revised manuscript.
We performed the lipidomic analyses under different conditions and the results were added as Figures 7C and 7D in the revised manuscript.Principal component analysis revealed significant differences in lipid composition between four samples.
We have added these results in the main text as follows: (Page 12, line 11-21, in the revised manuscript) "Subsequently, lipidomic analysis was then performed to determine whether the observed transcriptional changes in the phospholipid biosynthetic genes lead to the changes in the phospholipid composition in the cell.A principal component analysis was performed to understand the overall pattern of phospholipid variability in terms of each biological condition (Figure 7C).The results clustered into four groups, suggesting that lipid compositions differed among four sample sets.When we looked at alterations in the phospholipid composition more closely (Figure .7D), we found that PC, PE and PS contents in HGT1 Rim11 strain were significantly reduced by GSH treatment, which was consistent with decreases in the mRNA levels of OPI3, PSD1 and CHO1 as revealed by the RNA-seq analysis (Supplemental Figure 5B).In contrast, a PI content was increased upon GSH treatment.Moreover, RIM11 overexpression suppressed the reduction of PC, PE, and PS caused by GSH treatment and greatly increased the amount of PI.In contrast, overexpression of RIM11-K68A decreased suppressing effects on the reduction of these lipids.These results show that changes in the Rim11 kinase activity leads to changes in lipid compositions" As we performed the lipidomic analysis in our revision, we deleted the following sentence in the original manuscript in the Discussion.
Deleted sentence (Page 14, line 26 -28, in the original manuscript) "Lipidomic analysis of RIM11-overexpressing cells may help to better understand the mechanisms by which yeast cells respond to GSH stress and a wide range of other environmental stresses."Response: As suggested, our interpretation of the data regarding the test of RIM11 overexpression in the Δume6 HGT1 strain was incorrect.We corrected the main text as mentioned below.Additionally, as suggested, we created the Δrim11Δume6 HGT1 strain and tested its GSH stress sensitivity using a spot assay.We found that the Δrim11Δume6 HGT1 strain showed synthetic GSH sensitivity.We included this result as Figure 6D in the revised manuscript.This result suggests that Rim11 and Ume6 function in different pathways to cope with GSH stress.Based on these results, we revised the main text as follows.
(Page 10, line 23 -26, in the original manuscript) "In contrast, overexpression of RIM11 in the Δume6 HGT1 strain rendered it GSH stress resistant, although to a level lower than that of the HGT1 RIM11 OE strain.These results suggest that Rim11 substrates other than Ume6 may be involved in GSH stress tolerance, but that Ime1 may likely be ruled out as a potential candidate."(Page 11, line 11-13, in the revised manuscript) "In contrast, RIM11 overexpression in the Δume6 HGT1 strain only partially rescued the growth defect of the Δume6 HGT1 strain under GSH stress (Figure 6C).Furthermore, the Δrim11Δume6 HGT1 strain showed synthetic GSH sensitivity (Figure 6D), suggesting that Rim11 and Ume6 function in different pathways to cope with GSH stress." Reviewer #2 (Remarks to the Author): Yasukawa et al performed a screen designed to isolate genes that modulate the growth defect in S. cerevisiae associated with the uptake of high level of glutathione from the medium (facilitated by high-level expression of the plasma membrane glutathione transporter).They describe the isolation of three multicopy suppressors (RIM11, BHM1, and WHI2).Initially the authors focus on how loss or overexpression of these three genes impacts the ability of cells to survive under conditions of increased glutathione uptake.Later, the authors focus on Rim11 and explore how Rim11 kinase activity alters transcriptional profiling connected with the exposure to GSH.The authors propose a model for the upregulation of a subset of genes involved in lipid biogenesis that is dependent on Rim11 kinase activity and occurs in response to the cellular uptake of exogenous glutathione.The authors go on to make a series of observations about various players connected to Rim11 signaling and/or the transcription of lipid biogenesis genes, including OPI1, UME6, IME1, and INO1.They report transcriptional changes observed in the presence of GSH, as well as growth differences in the presence, absence, or overexpression of these genes.
Overall, the manuscript contains several interesting observations that suggest unexpected signaling activated in the presence of exogenous GSH, a less explored "stressor".Yet, in its current form, the manuscript feels incomplete in its goal to outline a clear Rim11-dependent signaling output that is associated with GSH uptake.While the authors outline a model in Figure 7, this model relies heavily on relationships anticipated based on prior studies focused on the signaling outlined in a variety of conditions, not necessarily connections established by the authors to occur as part of GSH-induced signaling.In broad terms, it feels like a significant jump from the reported data to the model of Figure 7, including proteins in the model that are not discussed or explored in the manuscript.The extent of the experiments do not seem to match with the overall conclusions and model.

Response:
We agree with the reviewer, and we have carefully revised the model figure (new Figure 8 in the revised manuscript) to reflect our data as accurately as possible.Moreover, we included Pah1, which plays an important role in the regulation of phospholipid biosynthesis and whose enzymatic activity is downregulated by phosphorylation by Rim11.We have addressed each of the specific comments below.

Some suggestions as to further studies that may help to focus or complete the study:
1. Arguably, the manuscript could benefit from some reorganization, with a clear focus from the initial results on Rim11.For example, reporting the initial isolation of RIM11 as a high-copy 2 suppressor, and a straightforward analysis of whether overexpression of RIM11 requires Ire1 to confer resistance to GSH in the HGT1 strain, would streamline the results.These straightforward experiments would establish a role for Rim11, and a role that is outside the prior focus on the UPR that the Toledano work reported in Kumar et al 2011.

Response:
We agree that reorganization may be meaningful.However, as we believe that discovery of two other suppressors is important for future studies on GSH stress, we hope to show the data we obtained regarding the relationship between the suppressors (Figure 1).We have carefully revised the main text related to Figure 1 to explain the results clearly as follows.
(Page 5-6, line 39-8, in the revised manuscript) "We further examined the GSH stress sensitivity of double or triple mutants of these three genes (Figure 1E).The Δbmh1Δwhi2 and Δrim11Δbmh1 HGT1 strains showed augmented GSH stress sensitivity compared with their single-deletion mutants, as shown in Fig. 1E.Furthermore, RIM11 overexpression did not rescue the GSH sensitivity of the Δbmh1 HGT1 strain, and BMH1 overexpression did not rescue the GSH sensitivity of Δrim11 HGT1 (Figure 1F).These results suggest that Bmh1 may function in a different pathway than Rim11.Overexpression of RIM11 partially rescued the GSH sensitivity of the Δwhi2 HGT1 strain.Also, overexpression of WHI2 rescued the GSH sensitivity of the Δrim11 HGT1 strain (Fig. 1F).These results suggest that Rim11 and Whi2 may function in the similar pathways.The Δwhi2Δrim11 HGT1 strain showed similar levels of sensitivity to Δrim11 HGT1 strain.In contrast, the Δwhi2 Δrim11 HGT1 strain was more sensitive to GSH than the Δwhi2 HGT1 strain (Fig. 1E).These results may suggest Rim11 has more important role than Whi2 in response to GSH stress." As per your suggestion, RIM11 was overexpressed on a high-copy plasmid in the Δire1 HGT1 strain and sensitivity to GSH stress was tested.We demonstrated that IRE1 is not necessary for RIM11 to function as a multicopy suppressor to GSH stress (new Figure 3D).This result strengthens our conclusion, which we have written as follows: "This result strongly suggests that Rim11 functions independently of the Ire1-dependent UPR".
GSH stress was tested.We found that IRE1 is not necessary for RIM11 to function as a multicopy suppressor of GSH stress (Figure 3D)." The figure legend (Figure 3; page 29, line 5-6, in the revised manuscript): "(D) RIM11 was overexpressed on a high-copy plasmid in the Δire1 HGT1 strain and sensitivity to GSH stress was tested."shows better growth than the whi2∆).This growth variability makes the argument as to whether there is a true additive impact, or if the difference in growth is just assay-to-assay variation, difficult to discern.Similarly, the growth suppression of the null strains with the various 2µ plasmids is suggestive, but not particularly robust or fully consistent with the outlined model.It is unclear in the outlined epistasis diagram why a bhm1∆ would not be bypassed by additional RIM11 (?) If the authors do want to make arguments about epistasis, some evidence of reproducibility is also warranted, including how many replicates were performed and/or showing replicates of the strains.Accordingly, the following underlined sentence was added to the main text.

Response
(Page 5, line 28-32 in the revised manuscript) "Deleting RIM11, BMH1, or WHI2 rendered the HGT1 strain sensitive to GSH stress.Introduction of BMH1 and WHI2 on a low-copy plasmid rescued the growth retardation phenotypes of the corresponding deletion mutants (Figure 1D).The ∆rim11 HGT1 strain showed slightly higher sensitivity to GSH than the Δwhi2 HGT1 strain, whereas the Δbmh1 HGT1 strain was less sensitive than the other two strains.Although the introduction of RIM11 under the control of its own promoter failed to rescue the growth phenotype of the Δrim11 HGT strain, " The lower panel of Figure 1D was also replaced with a figure that showed the results with improved clarity.
We agree that there was an overinterpretation of the data in the original manuscript.As we had already repeated the same experiments more than twice, the results are shown in the "Spot assays in Figure 1 were repeated more than twice and the results were reproducible." We avoided overinterpretation of the data and revised the manuscript to explain the results more accurately than in the original manuscript.We then carefully considered the spot assay results and concluded that it was difficult to create a diagram that reflected all the data correctly.Therefore, we have deleted the diagram in the revised manuscript.
We changed the main text as follows.
(Page 5-6, line 39-8, in the revised manuscript) "We further examined the GSH stress sensitivity of double or triple mutants of these three genes (Figure 1E).The Δbmh1Δwhi2 and Δrim11Δbmh1 HGT1 strains showed augmented GSH stress sensitivity compared with their single-deletion mutants, as shown in Fig. 1E.Furthermore, RIM11 overexpression did not rescue the GSH sensitivity of the Δbmh1 HGT1 strain, and BMH1 overexpression did not rescue the GSH sensitivity of Δrim11 HGT1 (Figure 1F).These results suggest that Bmh1 may function in a different pathway than Rim11.Overexpression of RIM11 partially rescued the GSH sensitivity of the Δwhi2 HGT1 strain.Also, overexpression of WHI2 rescued the GSH sensitivity of the Δrim11 HGT1 strain (Fig. 1F).These results suggest that Rim11 and Whi2 may function in the similar pathways.The Δwhi2Δrim11 HGT1 strain showed similar levels of sensitivity to Δrim11 HGT1 strain.In contrast, the Δwhi2 Δrim11 HGT1 strain was more sensitive to GSH than the Δwhi2 HGT1 strain (Fig. 1E).These results may suggest Rim11 has more important role than Whi2 in response to GSH stress." 3. The authors do not validate the transcriptional upregulation they outline for their RNA-seq dataset.Some validation by RT-PCR would strengthen the conclusions.It would also allow one to establish if Rim11 is required for the upregulation (e.g., analysis in a rim11∆).Such data would 4. The experiments of Figure 6 exploring Opi1, Ime1, Ume6 and Ino1 expression or localization during conditions of increased GSH are suggestive of unique roles.However, these data could be more robust.The variable expression of Opi1 in the "GSH stress" cells makes the co-localization data difficult to parse.

Response:
The ratio of cells with Opi1-GFP in the nucleus were counted and graphed.This graph was added to the revised manuscript as Figure 6B.As Opi1-GFP is expressed from the construct that is integrated in the chromosome, a reason for variable expression, as you suggested, is not clear.
However, we feel that the quantification of the nuclear localization upon GSH stress strengthens our original conclusion that GSH treatment induces nuclear localization of Opi1.We added this result in the revised manuscript as follows.
(Page 10-11, Line 39-2, in the revised manuscript): "The ratio of cells with GFP-Opi1 in the ER or nucleus were counted and graphed.Nuclear localization of GFP-Opi1 was significantly increased not only by glucose starvation, but also upon GSH treatment (Figure 6B and Supplemental Figure 6C), which suggests that Opi1 may be involved in the GSH stress response and represses the transcription of phospholipid biosynthetic genes." Moreover, there was a mistake in the original manuscript regarding GFP-OPI1 expression.In Figure 6B, GFP-OPI1 was introduced into the cell by homologous recombination, not on the plasmid.Accordingly, we deleted "harboring pRS316 empty vector" in the Figure 6B legend in the original manuscript as indicated below.
(Page 30; line 23-24, in the revised manuscript) "To determine the level of background fluorescence or autofluorescence originating from the live cells, images of HGT1 strain harboring pRS316 empty vector under GSH stress conditions were also acquired."

It is not clear why a more intense western band for Ume6-FLAG implies an increased in"Ume6 phosphorylation".
Response: To further test the possibility of phosphorylation of Ume6, we conducted experiments using biotinylated-Phos-tag and streptavidin-conjugated Alexa488.We immunoprecipitated 3xFLAG-Ume6, which was then separated by SDS-PAGE.Proteins were transferred to the membrane, which was probed with biotinylated-Phos-tag and streptavidin-conjugated Alexa488.A yeast protein known to be phosphorylated (whose identity we want to avoid revealing here because a manuscript is in preparation) was also included as a positive experimental control.Under various conditions, phosphorylation of positive control protein was always detected.However, phosphorylation of 3xFLAG-Ume6 was never clearly detected under the same experimental conditions.
Therefore, we concluded that there was a low possibility that Ume6 was sufficiently phosphorylated to be detected by the method we employed.Accordingly, we have deleted "Ume6 phosphorylation" (page 11, line 11 in the original manuscript).We thank you for bringing this to our attention.

The authors observe and say that the degree and/or location of phosphorylation on Rim11 is likely
distinct for GSH and glucose starvation, but yet then the authors argue that GSH induces a glucose starvation response (in the discussion).If the authors wish to imply that glucose transporters, etc. are important players in the response to GSH (as discussed), then some experiments to this point should be performed.

Response:
To analyze the behavior of glucose transporters in response to GSH addition, we created yeast strains in which there were glucose transporters to which GFP is C-terminally fused.Upon overexpression of RIM11, the protein abundance of Hxt1, a low-affinity glucose transporter, was decreased, and that of Hxt2, a high-affinity glucose transporter, was increased.It seems likely that this is one of the mechanisms by which RIM11 alleviates GSH stress as a high-copy suppressor.
GSH stress did not significantly change the protein abundance of either Hxt1-GFP or Hxt2-GFP.
However, there was a tendency for Hxt1-GFP to increase and a tendency for Hxt2-GFP to decrease (p = 0.059, n =3) upon GSH treatment.Furthermore, microscopic measurements revealed a significant increase of Hxt1-GFP signal at the plasma membrane upon GSH treatment.Results of the western blotting were included in Figure 6A and microscopic observations were included in Supplement Figure 6A and 6B in the revised manuscript.We also revised the main text as follows: (Page 10, line 15-30, in the revised manuscript) "…we first examined the regulatory factor involved in glucose sensing.During RIM11 OE-mediated GSH stress tolerance, a signal induced by the overexpressed RIM11 greatly increased the expression of MTH1 (log 2 fc of 2.42) via unknown mechanisms.In contrast, overexpression of RIM11 K68A substantially decreased (log 2 fc of -3.09) MTH1 expression (Figure 5D and Supplemental Table 2, see sample set [iii] and [iv]).Furthermore, the expression of the low-affinity glucose transporter HXT1 was repressed (log 2 fc of -3.12), whereas that of the high-affinity glucose transporter HXT2 was markedly upregulated (log 2 fc of 3.80) (Supplemental Table 2, see sample set [iv]).Considering that Mth1 also represses the expression of HXT1, a gene encoding a low-affinity glucose transporter, in the presence of high glucose (Roy et al., 2013), we hypothesized that a relationship may exist between the kinase activity of Rim1 and the dynamics of hexose transporters.To analyze the dynamics of glucose transporters in the GSH stress response, we created yeast strains producing glucose transporters to which GFP was fused c-terminally.RIM11 overexpression decreased the protein abundance of Hxt1, a low-affinity glucose transporter, and increased that of Hxt2, a high-affinity glucose transporter (Figure 6A).We hypothesized that this may be one of the mechanisms by which RIM11 alleviates GSH stress as a high-copy suppressor.We observed a tendency for Hxt1-GFP to increase and Hxt2-GFP to decrease (p = 0.059, n = 3) upon GSH treatment (Figure 6A).Moreover, microscopic measurements revealed a significant increase of Hxt1-GFP signal at the plasma membrane upon GSH treatment (Supplemental Figure 6A and 6B).
Therefore, these transcriptional and proteomic changes were presumed to alleviate the stress caused by low glucose." Or at a minimum, the transcriptional signature that the authors use in the discussion to support a glucose starvation response should be more clearly outlined in the results.
Response: As per your suggestion, we have relocated the sentence regarding Table S2 in 2, see sample set [iv]).Concomitantly, the expression of the low-affinity glucose transporter HXT1 was repressed (log 2 fc of -3.12), whereas that of the high-affinity glucose transporter HXT2 was markedly upregulated (log 2 fc of 3.80) (Supplemental Table 2, see sample set [iv]).Therefore, we believe that these transcriptional changes alleviate the stress caused by low-glucose levels and the transcriptional repression by Opi1." 6.The RNA-seq were completed after 2 h exposure to 250 µM GSH.The authors state that these data are "consistent with the results reported previously" of Kumar et al 2011.It seems a missed opportunity for a more descriptive comparison.Given the prior work followed the transcriptional response at an earlier and lower dose (50 µM GSH for 30 min), one would expect some differences.Thank you for submitting this revision, which has now been seen by the two original reviewers whose comments are found below.Both reviewers are supportive of publication, with a couple of minor changes suggested by reviewer 1.I'm happy to provisionally accept the study in advance of these changes.------------------------------------------------------------------------Dear Dr. Noda, The review of your manuscript, referenced above, is now complete.The Monitoring Editor has decided that your manuscript requires minor revisions before it can be published in Molecular Biology of the Cell, as described in the Monitoring Editor's decision letter above and the reviewer comments (if any) below.

Sincerely, Elizabeth Miller Monitoring Editor Molecular Biology of the Cell
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Sincerely, Eric Baker Journal Production Manager MBoC Editorial Office mbc@ascb.org------------------------------------------------------------------------Reviewer #1 (Remarks to the Author): This manuscript from Yusukawa et al., which is a revision of a previous manuscript, advances the understanding of the gluthatione stress response in yeast and gives insight into the significance of phospholipid metabolism in this response.This revision addresses most of the major concerns, including the lack of data showing that differentially expressed genes identified by RNA-seq are confirmed by examining protein levels, as well as the absence of data looking at cellular phospholipid content.Although most of the initial comments were adequately addressed, there remains an issue with Figure 8 8 in Khondker et al. for a recent review).The Rim11-mediated phosphorylation of Pah1 inhibits PA phosphatase activity.The inhibition of PA phosphatase activity by Rim11 would cause the accumulation of the substrate PA, the retention of Opi1 outside the nucleus, and the derepression of UAS INO -containing genes.Khondker et al (2022) also showed that cells expressing phosphorylation-deficient forms of Pah1 (T163A/T164A and S602A) exhibit an increase in the synthesis of triacylglycerol when Pah1 cannot be dephosphorylated by the Nem1-Spo7 complex.This is consistent with Pah1-mediated reduction in PA, translocation of Opi1 into the nucleus for repression of UAS INO -containing genes and increase in the synthesis of triacylglycerol at the expense of membrane phospholipids.Khondker et al. (2022) did not perform experiments that make the direct connection between Rim11 protein kinase activity and the transcription of UAS INO -containing genes, which is demonstrated in this study!Fig. 7 can be revised to show this connection.Response: Thank you for these helpful comments.We have addressed the points raised by clarifying the connections between GSH stress, functions of Rim11 and Pah1, and the change in Opi1 localization in a new model figure (Figure 8) in the revised manuscript.We have also included descriptions in the main text to explain the model in detail (see below, this letter Page 3, line 1 -29).

Figure 1E -
Figure 1E-bmh1Δ and whi2Δ show a synthetic growth defect when combined, which is in conflictwith the proposed model in Figure1Fthat shows them in the same pathway.

Figure 6B -
Figure 6B-Rim11 over expression only shows a partial rescue of the growth defect in ume6 deletion, not GSH stress resistance as stated in the text.This also doesn't show conclusively that Rim11 and Ume6 act in the same pathway in GSH stress response.This figure could benefit from a spot test that included strains in a rim11Δ background to determine if there are synthetic growth defects when combined with ume6Δ.
Figure 1E.These conclusions appear to be a potential overinterpretation of the data.It is also unclear what the pathway organization (if true) means in the context of the later analysis of, and focus on, Rim11.The growth defects observed between the three individual mutants on plates are modest and seem variable (Fig 1D the bhm1∆ and whi2∆ growth are called similar, versus in Fig 1E the bmh1∆

:
To address "Fig 1D the bhm1∆ and whi2∆ growth are called similar, versus in Fig 1E the bmh1∆ shows better growth than the whi2∆", we further tested the growth in the liquid medium and performed a spot assay.The results are shown in Figure for the reviewers B. These results confirmed that the Δwhi2 HGT1 strain was more sensitive to GSH than was the Δbmh1 HGT1 strain.
Figures for the reviewers C (replicates of Figure 1E in the main text) and in the Figures for the reviewers D (replicates of Figure 1F in the main text).In the Figures for the reviewers D, spot assays using at least three independent isolates in every combination were performed and the results were reproducible.Accordingly, we added the following sentence to the Figure 1 legend.(Page 28; Line 13-14, in the revised manuscript).
the Discussion to the Result section.The following two sentences have been relocated to the Results section: (Page 12, Line 33 in the original manuscript): Mth1 also represses the expression of HXT1, a gene encoding a low-affinity glucose transporter, in the presence of high glucose (Roy et al., 2013).(Page 12, Line 39 in the original manuscript) " During RIM11 OE-mediated GSH stress tolerance (Figure 7C), a signal induced by the overexpressed Rim11 greatly increased the expression of MTH1 (log 2 fc of 2.42) by unknown mechanisms (Supplemental Table

Response:
Figures for reviewers:

Fig. A :
Fig. A: Growth of the Δrim11 HGT1 strains carrying RIM11 with its upstream region (3,243 base) on the plasmid (CEN URA3) in the absence or presence of GSH treatment.Six clones were randomly chosen and their sensitivities to GSH-induced stress were evaluated using a spot test on SC-ura plates.Schematic diagram shows the cloned length of the upstream region of RIM11.Base 475 is an upstream region from the open reading frame of RIM11 in the Δrim11 HGT1 RIM11pr-RIM11 (CEN) strain displayed in the main Figure 1D.

Fig. B :
Fig. B: Comparison of the growth of the Δbmh1 HGT1 and Δwhi2 HGT1 strain with or without 50 µM GSH.Strains were cultured in SC without uracil at 30 o C. Spot test (upper) was performed with two independent experiments and a representative image is shown.During the growth time course (lower), cells (OD 600 of 0.1) were cultivated for 4 h and GSH was added.Line plots and error bars represent mean and standard deviation from three independent experiments (n = 9), respectively.Significant differences were analyzed using two-tailed Welch's t-test, ** p < 0.01, *** p < 0.001.

Fig. C :
Fig. C: Other independent repeated experiments related to Figure 1E in the main manuscript.Second and third replicates, different from that presented in FIGURE 1E, are shown.

Fig. D :
Fig. D: The number of biological replicates per sample with regards to Figure 1F in the main manuscript.
Table A: Logarithmic (base 2) values of the differentially expressed genes obtained from DNA microarray and RNA-seq.For the DNA microarray experiment, total RNA samples extracted from HGT1 and the Δrim11 HGT1 strains under GSH-induced stress (250 µM) were purified.cDNA from total RNA (200 ng) of each biological samples (n = 2) was labeled with Cys3, and subsequently hybridized to an 8 × 15 K Agilent microarray format (G4813A#16322, Yeast Saccharomyces cerevisiae).The transcript levels reflected the average values for each biological sample (n = 2) and are shown as the log 2 fc values.Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes" Dear Dr. Noda: Figure 8, which in panel A and C appear to show Rim11-dependent phosphorylation of Pah1 resulting in Pah1 translocation from the nuclear/ER membrane and being subject to proteasomal degradation.This model is inconsistent with Khondker et al. 2022.Although Khondker et al. showed that Rim11-dependent phosphorylation of Pah1 resulted in inhibition of catalytic activity, there was no significant on Pah1 localization or abundance, and thus this figure is misleading.I suggest that Rim11 be placed on the opposite arrow leading to the Nem1-Spo7 complex as shown in the Khondker et al paper.Response: Thank you for these helpful discussions.To accurately reflect the known roles of Pah1 phosphorylation, we have moved Rim11 in panel A and C in Figure 8 to the place close to the arrow leading to the Nem1-Spo7 complex as suggested.